+Open data
-Basic information
Entry | Database: PDB / ID: 7sbb | ||||||
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Title | Structure of type I-D Cascade bound to a ssRNA target | ||||||
Components |
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Keywords | RNA BINDING PROTEIN/RNA / CRISPR / Complex / Ribonucleoprotein complex / type I-D / type ID / type I / cyanobacteria / synechocystis / RNA binding protein / RNA BINDING PROTEIN-RNA complex | ||||||
Function / homology | Function and homology information CRISPR-associated protein Csc1 / CRISPR associated protein Csc3 / CRISPR-associated protein Csc2 / Csc2 Crispr Similarity search - Domain/homology | ||||||
Biological species | Synechocystis sp. PCC 6803 (bacteria) synthetic construct (others) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å | ||||||
Authors | Schwartz, E.A. / Taylor, D.W. | ||||||
Funding support | United States, 1items
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Citation | Journal: Nat Commun / Year: 2022 Title: Structural rearrangements allow nucleic acid discrimination by type I-D Cascade. Authors: Evan A Schwartz / Tess M McBride / Jack P K Bravo / Daniel Wrapp / Peter C Fineran / Robert D Fagerlund / David W Taylor / Abstract: CRISPR-Cas systems are adaptive immune systems that protect prokaryotes from foreign nucleic acids, such as bacteriophages. Two of the most prevalent CRISPR-Cas systems include type I and type III. ...CRISPR-Cas systems are adaptive immune systems that protect prokaryotes from foreign nucleic acids, such as bacteriophages. Two of the most prevalent CRISPR-Cas systems include type I and type III. Interestingly, the type I-D interference proteins contain characteristic features of both type I and type III systems. Here, we present the structures of type I-D Cascade bound to both a double-stranded (ds)DNA and a single-stranded (ss)RNA target at 2.9 and 3.1 Å, respectively. We show that type I-D Cascade is capable of specifically binding ssRNA and reveal how PAM recognition of dsDNA targets initiates long-range structural rearrangements that likely primes Cas10d for Cas3' binding and subsequent non-target strand DNA cleavage. These structures allow us to model how binding of the anti-CRISPR protein AcrID1 likely blocks target dsDNA binding via competitive inhibition of the DNA substrate engagement with the Cas10d active site. This work elucidates the unique mechanisms used by type I-D Cascade for discrimination of single-stranded and double stranded targets. Thus, our data supports a model for the hybrid nature of this complex with features of type III and type I systems. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7sbb.cif.gz | 634 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7sbb.ent.gz | 518.2 KB | Display | PDB format |
PDBx/mmJSON format | 7sbb.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7sbb_validation.pdf.gz | 795.7 KB | Display | wwPDB validaton report |
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Full document | 7sbb_full_validation.pdf.gz | 850.1 KB | Display | |
Data in XML | 7sbb_validation.xml.gz | 98.5 KB | Display | |
Data in CIF | 7sbb_validation.cif.gz | 155 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/sb/7sbb ftp://data.pdbj.org/pub/pdb/validation_reports/sb/7sbb | HTTPS FTP |
-Related structure data
Related structure data | 24976MC 7sbaC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Protein , 4 types, 11 molecules ABCDEFGHIJK
#1: Protein | Mass: 36527.109 Da / Num. of mol.: 7 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Gene: slr7012 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6ZEI6 #2: Protein | | Mass: 28942.748 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Gene: slr7013 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6ZEI5 #3: Protein | | Mass: 112067.508 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Gene: slr7011 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6ZEI7 #4: Protein | Mass: 17024.494 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Gene: slr7011 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6ZEI7 |
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-RNA chain , 2 types, 2 molecules XZ
#5: RNA chain | Mass: 10700.537 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
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#6: RNA chain | Mass: 13699.081 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Production host: Escherichia coli (E. coli) / References: GenBank: CP073020 |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Type I-D Cascade bound to a ssRNA target / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES | |||||||||||||||||||||||||
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Molecular weight | Value: 0.45 MDa / Experimental value: NO | |||||||||||||||||||||||||
Buffer solution | pH: 7.5 | |||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.16 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Monodisperse sample of elongated particles. | |||||||||||||||||||||||||
Specimen support | Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat | |||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K Details: Blot force 0, blot time 4s, no wait time between sample application and blotting. |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 22500 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 3 sec. / Electron dose: 41.2 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Particle selection | Num. of particles selected: 4100000 | ||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 167000 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | Details: Model was built de novo using coot then flexibly refined using ISOLDE. |